Blue white screening

Lisa Sam

1. S.Y.B.Sc . - PRACTICAL SESSION-3 (12 th Aug.2021) EXPERIMENT-2 EXPRESSION OF LAC EXPRESSION – BLUE WHITE SCREENING

2. EXPERIMENT-2 – STUDY of LAC EXPRESSION – BLUE WHITE SCREENING

3. Blue-White Screening & Protocols for Colony Selection IDENTIFICATION OF RECOMBINANT BACTERIA Blue-white screening is a rapid and efficient technique for the identification of recombinant bacteria. It relies on the activity of β-galactosidase, an enzyme occurring in  E. coli , which cleaves lactose into glucose and galactose.

4. The method is based on the principle of α-complementation of the  β-galactosidase  gene. This phenomenon of α-complementation was first demonstrated in work done by  Agnes Ullmann  in the laboratory of  François Jacob  and  Jacques Monod , where the function of an inactive mutant β-galactosidase with deleted sequence was shown to be rescued by a fragment of β-galactosidase in which that same sequence, the α-donor peptide, is still intact. [ Langley  et al.  showed that the mutant non-functional  β-galactosidase  was lacking in part of its N-terminus with its residues 11—41 deleted, but it may be complemented by a peptide formed of residues 3—90 of β-galactosidase. Blue-White Screening & Protocols for Colony Selection

5. DISRUPTING THE LACZ GENE The presence of lactose in the surrounding environment triggers the lacZ operon in  E. coli . The operon activity results in the production of β-galactosidase enzyme that metabolizes the lactose. Most plasmid vectors carry a short segment of lacZ gene that contains coding information for the first 146 amino acids of β-galactosidase. The host  E. coli  strains used are competent cells containing lacZΔM15 deletion mutation. When the plasmid vector is taken up by such cells, due to α-complementation process, a functional β-galactosidase enzyme is produced.

6. The plasmid vectors used in cloning are manipulated in such a way that this α-complementation process serves as a marker for recombination. A multiple cloning site (MCS) is present within the lacZ sequence in the plasmid vector. This sequence can be nicked by restriction enzymes to insert the foreign DNA. When a plasmid vector containing foreign DNA is taken up by the host  E. coli , the α-complementation does not occur, therefore, a functional β-galactosidase enzyme is not produced. If the foreign DNA is not inserted into the vector or if it is inserted at a location other than MCS, the lacZ gene in the plasmid vector complements the lacZ deletion mutation in the host  E. coli  producing a functional enzyme.

7. Video LINKS https://www.youtube.com/watch?v=Y7gxELssMRw https://www.youtube.com/watch?v=4fnS2xKjIbg